: Infection of humans with hepatitis B viruses cause acute and persistent infections of worldwide significance. Persistent infections, in particular, are associated with a very high risk of hepatocellular carcinoma (HCC). HCCs arise in HBV carriers after long periods of immunologically driven liver disease that is characterized by periods of inflammation, regeneration, cirrhosis and finally HCC. One of the hallmarks of HCCs arising in I-IBV carriers is the presence of clonally amplified viral DNA integrations. Our laboratory has carried out studies to understand the mechanisms by which viral DNA integrations may act as mutagenic agents during hepatocarcinogenesis. Our approach has been to study the natural history and frequency of integrations in clonal populations of cells. These studies have implicated a minor form of hepadnavirus DNA, specifically, double strand linear (DSL) viral DNA molecules as the precursors for integration and have also shown that DSL DNAs integrate at a higher frequency than wild type DNAs. Furthermmore, we have shown that integrations can be lost from the cells along with cellular DNA in a "hit and run" mutagenesis mechanism. In the current proposal we will utilize a new assay system that can detect single integrations to study integration frequencies of DSL versus WT viral DNA and follow the dynamic flux of integrations in cell culture. We will pursue integration studies during infection of primary hepatocytes under conditions that cause DNA damage and we will also utilize mutational analysis to test the hypothesis that topoisomerase I (top 1) acts as a regulator of viral replication and integration. These studies will increase our understanding of the genetic and physiologic controls of integration and their mutagenic impact on the host cell. Finally, in a new area of research, we will utilize cDNA microarrays to investigate the effects of the HBV X regualtory protein on the transcriptome of murine liver stem cells in both the liver progenitor and hepatocytic differentiation states. These studies will begin to help us understand the impact ofHBx on transcriptional regulation from a global perspective.